Vitamin D metabolite determination utilizing mass spectrometry following derivatization

ABSTRACT

The invention relates to the detection of vitamin D metabolites. In a particular aspect, the invention relates to methods for detecting derivatized vitamin D metabolites by mass spectrometry.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This application is a continuation of U.S. application Ser. No.13/287,012 filed Nov. 1, 2011, which is a continuation of U.S.application Ser. No. 13/115,935 filed May 25, 2011 (now U.S. Pat. No.8,076,157), which is a continuation of U.S. application Ser. No.12/630,790 filed Dec. 3, 2009 (now U.S. Pat. No. 7,977,117), each ofwhich is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the quantitative measurement of vitamin Dmetabolites. In a particular aspect, the invention relates to methodsfor quantitative measurement of vitamin D metabolites by HPLC-tandemmass spectrometry.

BACKGROUND OF THE INVENTION

Vitamin D is an essential nutrient with important physiological roles inthe positive regulation of calcium (Ca²⁺) homeostasis. Vitamin D can bemade de novo in the skin by exposure to sunlight or it can be absorbedfrom the diet. There are two forms of vitamin D; vitamin D₂(ergocalciferol) and vitamin D₃ (cholecalciferol). Vitamin D₃ is theform synthesized de novo by animals. It is also a common supplementadded to milk products and certain food products produced in the UnitedStates. Both dietary and intrinsically synthesized vitamin D₃ mustundergo metabolic activation to generate the bioactive metabolites. Inhumans, the initial step of vitamin D₃ activation occurs primarily inthe liver and involves hydroxylation to form the intermediate metabolite25-hydroxycholecalciferol (calcifediol; 25OHD₃). Calcifediol is themajor form of Vitamin D₃ in circulation. Circulating 25OHD₃ is thenconverted by the kidney to form 1α,25-dihydroxyvitamin D₃ (calcitriol;1α,25(OH)₂D₃), which is generally believed to be the metabolite ofVitamin D₃ with the highest biological activity.

Vitamin D₂ is derived from fungal and plant sources. Manyover-the-counter dietary supplements contain ergocalciferol (vitamin D₂)rather than cholecalciferol (vitamin D₃). Drisdol, the only high-potencyprescription form of vitamin D available in the United States, isformulated with ergocalciferol. Vitamin D₂ undergoes a similar pathwayof metabolic activation in humans as Vitamin D₃, forming the metabolites25OHD₂ and 1α,25(OH)₂D₂. Vitamin D₂ and vitamin D₃ have long beenassumed to be biologically equivalent in humans, however recent reportssuggest that there may be differences in the bioactivity andbioavailability of these two forms of vitamin D (Armas et. al., (2004)J. Clin. Endocrinol. Metab. 89:5387-5391).

Measurement of vitamin D, the inactive vitamin D precursor, is rare inclinical settings. Rather, serum levels of 25-hydroxyvitamin D₃,25-hydroxyvitamin D₂, and total 25-hydroxyvitamin D (“25OHD”) are usefulindices of vitamin D nutritional status and the efficacy of certainvitamin D analogs. The measurement of 25OHD is commonly used in thediagnosis and management of disorders of calcium metabolism. In thisrespect, low levels of 25OHD are indicative of vitamin D deficiencyassociated with diseases such as hypocalcemia, hypophosphatemia,secondary hyperparathyroidism, elevated alkaline phosphatase,osteomalacia in adults and rickets in children. In patients suspected ofvitamin D intoxication, elevated levels of 25OHD distinguishes thisdisorder from other disorders that cause hypercalcemia.

Measurement of 1α,25(OH)₂D is also used in clinical settings. Certaindisease states can be reflected by circulating levels of 1α,25(OH)₂D,for example kidney disease and kidney failure often result in low levelsof 1α,25(OH)₂D. Elevated levels of 1,25(OH)₂D may be indicative ofexcess parathyroid hormone or can be indicative of certain diseases suchas sarcoidosis or certain types of lymphomas.

Detection of vitamin D metabolites has been accomplished byradioimmunoassay with antibodies co-specific for 25OHD₂ and 25OHD₃.Because the current immunologically-based assays do not separatelyresolve 25OHD₂ and 25OHD₃, the source of any nutritional deficiency ofvitamin D cannot be determined without resorting to other tests. Reportshave been published that disclose methods for detecting specific vitaminD metabolites using mass spectrometry. In some of the reports, thevitamin D metabolites are derivatized prior to mass spectrometry, but inothers, they are not. For example Holmquist, et al., U.S. patentapplication Ser. No. 11/946,765, filed Dec. 28, 2007; Yeung B, et al., JChromatogr. 1993, 645(1):115-23; Higashi T, et al., Steroids. 2000,65(5):281-94; Higashi T, et al., Biol Pharm Bull. 2001, 24(7):738-43;Higashi T, et al., J Pharm Biomed Anal. 2002, 29(5):947-55; Higashi T,et al., Anal. Biochanal Chem, 2008, 391:229-38; and Aronov, et al., AnalBioanal Chem, 2008, 391:1917-30 disclose methods for detecting variousvitamin D metabolites by derivatizing the metabolites prior to massspectrometry. Methods to detect underivatized vitamin D metabolites arereported in Clarke, et al., in U.S. patent application Ser. No.11/101,166, filed Apr. 6, 2005, and Ser. No. 11/386,215, filed Mar. 21,2006, and Singh, et al., in U.S. patent application Ser. No. 10/977,121,filed Oct. 24, 2004.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the amount of oneor more vitamin D metabolites in a sample by mass spectrometry,including tandem mass spectrometry. The methods include derivatizingvitamin D metabolites in the sample prior to analysis.

In one aspect, the invention provides methods for determining the amountof one or more vitamin D metabolites in a sample by mass spectrometry.In some embodiments, the methods include the steps of: (i) subjectingone or more 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD)-derivatizedvitamin D metabolites in the sample to an extraction column and ananalytical column; (ii) subjecting one or more PTAD-derivatized vitaminD metabolites in the sample to an ionization source to generate one ormore ions detectable by mass spectrometry; (iii) determining the amountof one or more of the PTAD-derivatized vitamin D metabolite ions by massspectrometry; and (iv) relating the amount of PTAD-derivatized vitamin Dmetabolite ions determined in step (iii) to the amount of a vitamin Dmetabolite in the sample. In these methods, the sample is subjected to4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) under conditions sufficientto generate one or more PTAD-derivatized vitamin D metabolites prior tostep (i). In some embodiments, the sample is subject to turbulent flowliquid chromatography (TFLC) as the extraction column, and no analyticalcolumn is used. In other embodiments, no chromatography, includingextraction chromatography, is used. In these methods, the derivatizedvitamin D metabolites may be ionized with laser diode thermal desorption(LDTD).

In some embodiments, the extraction column of step (i) is a solid-phaseextraction (SPE) column; preferably, the extraction column of step (i)is a turbulent flow liquid chromatography (TFLC) column. In someembodiments, the analytical column of step (i) is a high performanceliquid chromatography (HPLC) column.

In some embodiments, the methods include the steps of: (i) subjectingthe sample to turbulent flow liquid chromatography (TFLC); (ii) ionizingone or more Cookson-type vitamin D metabolite derivatives in the sampleto generate one or more ions detectable by mass spectrometry; (iii)determining the amount of one or more of the Cookson-type vitamin Dmetabolite derivative ions by mass spectrometry; and (iv) relating theamount of Cookson-type vitamin D metabolite derivative ions determinedin step (iv) to the amount of a vitamin D metabolite in the sample. Inthese embodiments, the sample is subjected to a Cookson-typederivatizing reagent under conditions sufficient to generate one or moreCookson-type vitamin D metabolite derivatives prior to step (i). In someembodiments, the sample is subjected to high performance liquidchromatography (HPLC) after step (i) but prior to step (ii). In somerelated embodiments, the TFLC of step (i), the HPLC, and the ionizationof step (ii) are conducted in an on-line fashion.

Cookson-type derivatizing reagents useful for these methods may beselected from the group consisting of4-phenyl-1,2,4-triazoline-3,5-dione (PTAD),4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione(DMEQTAD), 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD), and4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), and isotopicallylabeled variants thereof. In preferred embodiments, the Cookson-typederivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) or anisotopically labeled variant thereof.

In some embodiments, the methods include the steps of: (i) subjectingthe sample to 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) underconditions sufficient to generate one or more PTAD-vitamin D metabolitederivatives; (ii) ionizing one or more PTAD-vitamin D metabolitederivatives in the sample with laser diode thermal desorption togenerate one or more ions detectable by mass spectrometry; (iii)determining the amount of one or more of the PTAD-vitamin D metabolitederivative ions by mass spectrometry; and (v) relating the amount ofPTAD-vitamin D metabolite derivative ions determined in step (iv) to theamount of a vitamin D metabolite in the sample; wherein the one or morevitamin D metabolite derivatives are selected from the group consistingof 25-hydroxyvitamin D₂ (25OHD₂), 25-hydroxyvitamin D₃ (25OHD₃),1α,25-dihydroxyvitamin D₂ (1α,25(OH)₂D₂), and 1α,25-dihydroxyvitamin D₃(1α,25(OH)₂D₃). In some embodiments, the one or more vitamin Dmetabolite derivatives comprise two or more selected from the groupconsisting of 25-hydroxyvitamin D₂ (25OHD₂), 25-hydroxyvitamin D₃(25OHD₃), 1α,25-dihydroxyvitamin D₂ (1α,25(OH)₂D₂), and1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃). In related embodiments, thetwo or more vitamin D metabolite derivatives are ionized simultaneously.In some embodiments, the sample is not subjected to chromatography. Insome embodiments, the sample is not subjected to liquid chromatography.

In the methods described herein, mass spectrometry may be tandem massspectrometry. In embodiments utilizing tandem mass spectrometry, tandemmass spectrometry may be conducted by any method known in the art,including for example, multiple reaction monitoring, precursor ionscanning, or product ion scanning.

In the methods described herein, the one or more vitamin D metabolitesmay comprise one or more vitamin D metabolites selected from the groupconsisting of 25-hydroxyvitamin D₂ (25OHD₂), 25-hydroxyvitamin D₃(25OHD₃), 1α,25-dihydroxyvitamin D₂ (1α,25(OH)₂D₂), and1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃); such as comprising25-hydroxyvitamin D₂ (25OHD₂) and 25-hydroxyvitamin D₃ (25OHD₃), orcomprising 1α,25-dihydroxyvitamin D₂ (1α,25(OH)₂D₂) and1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃).

Thus, in some embodiments, the one or more Cookson-type derivatizedvitamin D metabolites, or the one or more PTAD-derivatized vitamin Dmetabolites, comprise one or more of the group consisting ofPTAD-derivatized 25-hydroxyvitamin D₂ (PTAD-25OHD₂), PTAD-derivatized25-hydroxyvitamin D₃ (PTAD-25OHD₃), PTAD-derivatized1α,25-dihydroxyvitamin D₂ (PTAD-1α,25(OH)₂D₂), and PTAD-derivatized1α,25-dihydroxyvitamin D₃ (PTAD-1α,25(OH)₂D₃).

In embodiments where the one or more Cookson-type derivatized vitamin Dmetabolites, or the one or more PTAD-derivatized vitamin D metabolites,comprise PTAD-derivatized 25-hydroxyvitamin D₂ (PTAD-25OHD₂), the one ormore Cookson-type or PTAD-derivatized vitamin D metabolite ions compriseions selected from the group consisting of ions with a mass/charge ratio(m/z) of 570.3±0.5 and 298.1±0.5. In some embodiments, the one or moreCookson-type or PTAD-derivatized vitamin D metabolite ions comprise aparent ion with a mass/charge ratio (m/z) of 570.3±0.5 and a fragmention with a mass/charge ratio (m/z) of 298.1±0.5.

In embodiments where the one or more Cookson-type derivatized vitamin Dmetabolites, or the one or more PTAD-derivatized vitamin D metabolites,comprise PTAD-derivatized 25-hydroxyvitamin D₃ (PTAD-25OHD₃), the one ormore Cookson-type or PTAD-derivatized vitamin D metabolite ions compriseions selected from the group consisting of ions with a mass/charge ratio(m/z) of 558.3±0.5 and 298.1±0.5. In some embodiments, the one or moreCookson-type or PTAD-derivatized vitamin D metabolite ions comprise aparent ion with a mass/charge ratio (m/z) of 558.3±0.5 and a fragmention with a mass/charge ratio (m/z) of 298.1±0.5.

In embodiments where the one or more Cookson-type derivatized vitamin Dmetabolites, or the one or more PTAD-derivatized vitamin D metabolites,comprise PTAD-derivatized 1α,25-dihydroxyvitamin D₂ (PTAD-1α,25(OH)₂D₂),the one or more Cookson-type or PTAD-derivatized vitamin D metaboliteions comprise ions selected from the group consisting of ions with amass/charge ratio (m/z) of 550.4±0.5, 568.4±0.5, 277.9±0.5, and298.0±0.5. In some embodiments, the one or more Cookson-type orPTAD-derivatized vitamin D metabolite ions comprise a parent ion with amass/charge ratio (m/z) of 550.4±0.5 and a fragment ion with amass/charge ratio (m/z) of 277.9±0.5. In some embodiments, the one ormore Cookson-type or PTAD-derivatized vitamin D metabolite ions comprisea parent ion with a mass/charge ratio (m/z) of 568.4±0.5 and a fragmention with a mass/charge ratio (m/z) of 298.0±0.5.

In embodiments where the one or more Cookson-type derivatized vitamin Dmetabolites, or the one or more PTAD-derivatized vitamin D metabolites,comprise PTAD-derivatized 1α,25-dihydroxyvitamin D₃ (PTAD-1α,25(OH)₂D₃),the one or more Cookson-type or PTAD-derivatized vitamin D metaboliteions comprise ions selected from the group consisting of ions with amass/charge ratio (m/z) of 538.4±0.5, 556.4±0.5, 278.1±0.5, and298.0±0.5. In some embodiments, the one or more Cookson-type orPTAD-derivatized vitamin D metabolite ions comprise a parent ion with amass/charge ratio (m/z) of 538.4±0.5 and a fragment ion with amass/charge ratio (m/z) of 278.1±0.5. In some embodiments, the one ormore Cookson-type or PTAD-derivatized vitamin D metabolite ions comprisea parent ion with a mass/charge ratio (m/z) of 556.4±0.5 and a fragmention with a mass/charge ratio (m/z) of 298.0±0.5.

In embodiments which utilize two or more of an extraction column, ananalytical column, and an ionization source, two or more of thesecomponents may be connected in an online fashion to allow for automatedsample processing and analysis.

As used herein, the term “vitamin D metabolite” refers to any vitamin Danalog or any chemical species related to vitamin D generated by abiotransformation of vitamin D, such as intermediates and products ofvitamin D metabolism. Vitamin D metabolites may include chemical speciesgenerated by biotransformation of analogs of, or a chemical speciesrelated to, vitamin D₂ or vitamin D₃. Vitamin D metabolites may be foundin the circulation of an animal and/or may be generated by a biologicalorganism, such as an animal. Vitamin D metabolites may be metabolites ofnaturally occurring forms of vitamin D or may be metabolites ofsynthetic vitamin D analogs. In certain embodiments a vitamin Dmetabolite is one or more compounds selected from the group consistingof 25-hydroxyvitamin D₃, 25-hydroxyvitamin D₂, 1α,25-dihydroxyvitamin D₃and 1α,25-dihydroxyvitamin D₂.

As used herein, “derivatizing” means reacting two molecules to form anew molecule. Thus, a derivatizing agent is an agent that may be reactedwith another substance to derivatize the substance. For example,4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is a derivatizing reagentthat may be reacted with a vitamin D metabolite to form aPTAD-derivatized vitamin D metabolite.

As used here, the names of derivatized forms vitamin D metabolitesinclude an indication as to the nature of derivatization. For example,the PTAD derivative of 25-hydroxyvitamin D₂ is indicated asPTAD-25-hydroxyvitamin D₂ (or PTAD-25OHD₂).

As used herein, a “Cookson-type derivatizing agent” is a 4-substituted1,2,4-triazoline-3,5-dione compound. Exemplary Cookson-type derivatizingagents include 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD),4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione(DMEQTAD), 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD), and4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD). Additionally,isotopically labeled variants of Cookson-type derivatizing agents may beused in some embodiments. For example, the ¹³C₆-PTAD isotopic variant is6 mass units heavier than normal PTAD and may be used in someembodiments. Derivatization of vitamin D metabolites by Cookson-typereagents can be conducted by any appropriate method. See, e.g.,Holmquist, et al., U.S. patent application Ser. No. 11/946,765, filedDec. 28, 2007; Yeung B, et al., J. Chromatogr. 1993, 645(1):115-23;Higashi T, et al., Steroids. 2000, 65(5):281-94; Higashi T, et al., BiolPharm Bull. 2001, 24(7):738-43; Higashi T, et al., J Pharm Biomed Anal.2002, 29(5):947-55; Higashi T, et al., Anal. Biochanal Chem, 2008,391:229-38; and Aronov, et al., Anal Bioanal Chem, 2008, 391:1917-30.

In certain preferred embodiments of the methods disclosed herein, massspectrometry is performed in positive ion mode. Alternatively, massspectrometry is performed in negative ion mode. Various ionizationsources, including for example atmospheric pressure chemical ionization(APCI), laser diode thermal desorption (LDTD), or electrosprayionization (ESI), may be used in embodiments of the present invention.In certain preferred embodiments, vitamin D metabolites are measuredusing APCI or LDTD in positive ion mode.

In preferred embodiments, one or more separately detectable internalstandards are provided in the sample, the amount of which are alsodetermined in the sample. In these embodiments, all or a portion of boththe analyte(s) of interest and the internal standard(s) present in thesample are ionized to produce a plurality of ions detectable in a massspectrometer, and one or more ions produced from each are detected bymass spectrometry. Preferably, the internal standard(s) are one or moreof 25OHD₂-[6, 19, 19]-²H₃, 25OHD₂-[26, 26, 26, 27, 27, 27]-²H₆,25OHD₃-[6, 19, 19]-²H₃, 25OHD₃-[26, 26, 26, 27, 27, 27]-²H₆,1α,25(OH)₂D₂-[6, 19, 19]-²H₃, 1α,25(OH)₂D₂-[26, 26, 26, 27, 27, 27]-²H₆,1α,25(OH)₂D₃-[6, 19, 19]-²H₃, and 1α,25(OH)₂D₃-[26, 26, 26, 27, 27,27]-²H₆.

One or more separately detectable internal standards may be provided inthe sample prior to treatment of the sample with a Cookson-typederivatizing reagent. In these embodiments, the one or more internalstandards may undergo derivatization along with the endogenous vitamin Dmetabolites, in which case ions of the derivatized internal standardsare detected by mass spectrometry. In these embodiments, the presence oramount of ions generated from the analyte of interest may be related tothe presence or amount of analyte of interest in the sample. In someembodiments, the internal standards may be isotopically labeled versionsof vitamin D metabolites, such as 25OHD₂-[6, 19, 19]-²H₃, 25OHD₂-[26,26, 26, 27, 27, 27]-²H₆, 25OHD₃-[6, 19, 19]-²H₃, 25OHD₃-[26, 26, 26, 27,27, 27]-²H₆, 1α,25(OH)₂D₂-[6, 19, 19]-²H₃, 1α,25(OH)₂D₂-[26, 26, 26, 27,27, 27]-²H₆, 1α,25(OH)₂D₃-[6, 19, 19]-²H₃, and 1α,25(OH)₂D₃-[26, 26, 26,27, 27, 27]-²H₆.

Ions detectable in a mass spectrometer may be generated for each of theabove identified exemplary internal standards, as demonstrated inExamples 14 and 15, and FIGS. 7-8, 10-11, 13, and 15-16.

As used herein, an “isotopic label” produces a mass shift in the labeledmolecule relative to the unlabeled molecule when analyzed by massspectrometric techniques. Examples of suitable labels include deuterium(²H), ¹³C, and ¹⁵N. For example, 25OHD₂-[6, 19, 19]-²H₃ and 25OHD₃-[6,19, 19]-²H₃ have masses about 3 mass units higher than native 25OHD₂ and25OHD₃. The isotopic label can be incorporated at one or more positionsin the molecule and one or more kinds of isotopic labels can be used onthe same isotopically labeled molecule.

In other embodiments, the amount of the vitamin D metabolite ion or ionsmay be determined by comparison to one or more external referencestandards. Exemplary external reference standards include blank plasmaor serum spiked with one or more of 25OHD₂-[6, 19, 19]-²H₃, 25OHD₂-[26,26, 26, 27, 27, 27]-²H₆, 25OHD₃-[6, 19, 19]-²H₃, 25OHD₃-[26, 26, 26, 27,27, 27]-²H₆, 1α,25(OH)₂D₂-[6, 19, 19]-²H₃, 1α,25(OH)₂D₂-[26, 26, 26, 27,27, 27]-²H₆, 1α,25(OH)₂D₃-[6, 19, 19]-²H₃, and 1α,25(OH)₂D₃-[26, 26, 26,27, 27, 27]-²H₆. External standards typically will undergo the sametreatment and analysis as any other sample to be analyzed, includingtreatment with one or more Cookson-type reagents prior to massspectrometry.

In certain preferred embodiments, the limit of quantitation (LOQ) of25OHD₂ is within the range of 1.9 ng/mL to 10 ng/mL, inclusive;preferably within the range of 1.9 ng/mL to 5 ng/mL, inclusive;preferably about 1.9 ng/mL. In certain preferred embodiments, the limitof quantitation (LOQ) of 25OHD₃ is within the range of 3.3 ng/mL to 10ng/mL, inclusive; preferably within the range of 3.3 ng/mL to 5 ng/mL,inclusive; preferably about 3.3 ng/mL.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedparent or daughter ions by mass spectrometry. Relative reduction as thisterm is used does not require that any substance, present with theanalyte of interest in the material to be purified, is entirely removedby purification.

As used herein, the term “solid phase extraction” or “SPE” refers to aprocess in which a chemical mixture is separated into components as aresult of the affinity of components dissolved or suspended in asolution (i.e., mobile phase) for a solid through or around which thesolution is passed (i.e., solid phase). In some instances, as the mobilephase passes through or around the solid phase, undesired components ofthe mobile phase may be retained by the solid phase resulting in apurification of the analyte in the mobile phase. In other instances, theanalyte may be retained by the solid phase, allowing undesiredcomponents of the mobile phase to pass through or around the solidphase. In these instances, a second mobile phase is then used to elutethe retained analyte off of the solid phase for further processing oranalysis. SPE, including TFLC, may operate via a unitary or mixed modemechanism. Mixed mode mechanisms utilize ion exchange and hydrophobicretention in the same column; for example, the solid phase of amixed-mode SPE column may exhibit strong anion exchange and hydrophobicretention; or may exhibit column exhibit strong cation exchange andhydrophobic retention.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and turbulent flow liquidchromatography (TFLC) (sometimes known as high turbulence liquidchromatography (HTLC) or high throughput liquid chromatography).

As used herein, the term “high performance liquid chromatography” or“HPLC” (sometimes known as “high pressure liquid chromatography”) refersto liquid chromatography in which the degree of separation is increasedby forcing the mobile phase under pressure through a stationary phase,typically a densely packed column.

As used herein, the term “turbulent flow liquid chromatography” or“TFLC” (sometimes known as high turbulence liquid chromatography or highthroughput liquid chromatography) refers to a form of chromatographythat utilizes turbulent flow of the material being assayed through thecolumn packing as the basis for performing the separation. TFLC has beenapplied in the preparation of samples containing two unnamed drugs priorto analysis by mass spectrometry. See, e.g., Zimmer et al., J ChromatogrA 854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368,5,795,469, and 5,772,874, which further explain TFLC. Persons ofordinary skill in the art understand “turbulent flow”. When fluid flowsslowly and smoothly, the flow is called “laminar flow”. For example,fluid moving through an HPLC column at low flow rates is laminar. Inlaminar flow the motion of the particles of fluid is orderly withparticles moving generally in straight lines. At faster velocities, theinertia of the water overcomes fluid frictional forces and turbulentflow results. Fluid not in contact with the irregular boundary “outruns”that which is slowed by friction or deflected by an uneven surface. Whena fluid is flowing turbulently, it flows in eddies and whirls (orvortices), with more “drag” than when the flow is laminar. Manyreferences are available for assisting in determining when fluid flow islaminar or turbulent (e.g., Turbulent Flow Analysis: Measurement andPrediction, P. S. Bernard & J. M. Wallace, John Wiley & Sons, Inc.,(2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott,Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 50 μm.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. In a preferredembodiment the analytical column contains particles of about 5 μm indiameter. Such columns are often distinguished from “extractioncolumns”, which have the general purpose of separating or extractingretained material from non-retained materials in order to obtain apurified sample for further analysis.

As used herein, the terms “on-line” and “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrometric instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500,entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat.No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;”U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced PhotolabileAttachment And Release For Desorption And Detection Of Analytes;” Wrightet al., Prostate Cancer and Prostatic Diseases 1999, 2: 264-76; andMerchant and Weinberger, Electrophoresis 2000, 21: 1164-67.

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photoionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber, which is heated slightly to prevent condensation and toevaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Anal. Chem.2000, 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase. Laser diode thermal desorption (LDTD) is a technique wherein asample containing the analyte is thermally desorbed into the gas phaseby a laser pulse. The laser hits the back of a specially made 96-wellplate with a metal base. The laser pulse heats the base and the heatcauses the sample to transfer into the gas phase. The gas phase samplemay then be drawn into an ionization source, where the gas phase sampleis ionized in preparation for analysis in the mass spectrometer. Whenusing LDTD, ionization of the gas phase sample may be accomplished byany suitable technique known in the art, such as by ionization with acorona discharge (for example by APCI).

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, the term “lower limit of quantification”, “lower limitof quantitation” or “LLOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a relative standarddeviation (RSD %) of less than 20% and an accuracy of 80% to 120%.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as three times the RSD ofthe mean at the zero concentration.

As used herein, an “amount” of an analyte in a body fluid sample refersgenerally to an absolute value reflecting the mass of the analytedetectable in volume of sample. However, an amount also contemplates arelative amount in comparison to another analyte amount. For example, anamount of an analyte in a sample can be an amount which is greater thana control or normal level of the analyte normally present in the sample.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D show exemplary chromatograms for 250H-D₃-PTAD, [6, 19,19]-²H₃-25OHD₃-PTAD (internal standard), 25OHD₂-PTAD, and [6, 19,19]-²H₃-25OHD₂-PTAD (internal standard), respectively. Details arediscussed in Example 3.

FIGS. 2A and 2B show exemplary calibration curves for 25OHD₂ and 25OHD₃in serum samples determined by methods described in Example 3.

FIG. 3A shows a plots of coefficient of variation versus concentrationfor 25OHD₂ and 25OHD₃. FIG. 3B shows the same plot expanded near theLLOQ. Details are described in Example 4.

FIGS. 4A-B show linear regression and Deming regression analyses for thecomparison of mass spectrometric determination of 25OHD₂ with andwithout PTAD derivatization. Details are described in Example 10.

FIGS. 5A-B show linear regression and Deming regression analyses for thecomparison of mass spectrometric determination of 25OHD₃ with andwithout PTAD derivatization. Details are described in Example 10.

FIG. 6A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 520 to 620) for PTAD-25-hydroxyvitamin D₂ ions. FIG. 6B shows anexemplary product ion spectra (covering the m/z range of about 200 to400) for fragmentation of the PTAD-25-hydroxyvitamin D₂ precursor ionwith m/z of about 570.3. Details are described in Example 14.

FIG. 7A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 520 to 620) for PTAD-25-hydroxyvitamin D₂-[6, 19, 19]-²H₃ ions.FIG. 7B shows an exemplary product ion spectra (covering the m/z rangeof about 200 to 400) for fragmentation of the PTAD-25-hydroxyvitaminD₂-[6, 19, 19]-²H₃ precursor ion with m/z of about 573.3. Details aredescribed in Example 14.

FIG. 8A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 520 to 620) for PTAD-25-hydroxyvitamin D₂-[26, 26, 26, 27, 27,27]-²H₆ ions. FIG. 8B shows an exemplary product ion spectra (coveringthe m/z range of about 200 to 400) for fragmentation of thePTAD-25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆ precursor ionwith m/z of about 576.3. Details are described in Example 14.

FIG. 9A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 520 to 620) for PTAD-25-hydroxyvitamin D₃ ions. FIG. 9B shows anexemplary product ion spectra (covering the m/z range of about 200 to400) for fragmentation of the PTAD-25-hydroxyvitamin D₃ precursor ionwith m/z of about 558.3. Details are described in Example 14.

FIG. 10A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 520 to 620) for PTAD-25-hydroxyvitamin D₃-[6, 19, 19]-²H₃ ions.FIG. 10B shows an exemplary product ion spectra (covering the m/z rangeof about 200 to 400) for fragmentation of the PTAD-25-hydroxyvitaminD₃-[6, 19, 19]-²H₃ precursor ion with m/z of about 561.3. Details aredescribed in Example 14.

FIG. 11A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 520 to 620) for PTAD-25-hydroxyvitamin D₃-[26, 26, 26, 27, 27,27]-²H₆ ions. FIG. 11B shows an exemplary product ion spectra (coveringthe m/z range of about 200 to 400) for fragmentation of thePTAD-25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ precursor ionwith m/z of about 564.3. Details are described in Example 14.

FIG. 12A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 520 to 620) for PTAD-1α,25-dihydroxyvitamin D₂ ions. FIG. 12Bshows an exemplary product ion spectra (covering the m/z range of about250 to 350) for fragmentation of the PTAD-1α,25-dihydroxyvitamin D₂precursor ion with m/z of about 550.4. FIG. 12C shows an exemplaryproduct ion spectra (covering the m/z range of about 250 to 350) forfragmentation of the PTAD-1α,25-dihydroxyvitamin D₂ precursor ion withm/z of about 568.4. FIG. 12D shows an exemplary product ion spectra(covering the m/z range of about 250 to 350) for fragmentation of thePTAD-1α,25-dihydroxyvitamin D₂ precursor ion with m/z of about 586.4.Details are described in Example 15.

FIG. 13A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 520 to 620) for PTAD-1α,25-dihydroxyvitamin D₂-[26, 26, 26, 27,27, 27]-²H₆ ions. FIG. 13B shows an exemplary product ion spectra(covering the m/z range of about 250 to 350) for fragmentation of thePTAD-1α,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆ precursorion with m/z of about 556.4. FIG. 13C shows an exemplary product ionspectra (covering the m/z range of about 250 to 350) for fragmentationof the PTAD-1α,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆precursor ion with m/z of about 574.4 FIG. 13D shows an exemplaryproduct ion spectra (covering the m/z range of about 250 to 350) forfragmentation of the PTAD-1α,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27,27]-²H₆ precursor ion with m/z of about 592.4. Details are described inExample 15.

FIG. 14A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 520 to 620) for PTAD-1α,25-hydroxyvitamin D₃ ions. FIG. 14B showsan exemplary product ion spectra (covering the m/z range of about 250 to350) for fragmentation of the PTAD-1α,25-dihydroxyvitamin D₃ precursorion with m/z of about 538.4. FIG. 14C shows an exemplary product ionspectra (covering the m/z range of about 250 to 350) for fragmentationof the PTAD-1α,25-dihydroxyvitamin D₃ precursor ion with m/z of about556.4. FIG. 14D shows an exemplary product ion spectra (covering the m/zrange of about 250 to 350) for fragmentation of thePTAD-1α,25-dihydroxyvitamin D₃ precursor ion with m/z of about 574.4.Details are described in Example 15.

FIG. 15A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 520 to 620) for PTAD-1α,25-dihydroxyvitamin D₃-[6, 19, 19]-²H₃ions. FIG. 15B shows an exemplary product ion spectra (covering the m/zrange of about 250 to 350) for fragmentation of thePTAD-1α,25-dihydroxyvitamin D₃-[6, 19, 19]-²H₃ precursor ion with m/z ofabout 541.4. FIG. 15C shows an exemplary product ion spectra (coveringthe m/z range of about 250 to 350) for fragmentation of thePTAD-1α,25-dihydroxyvitamin D₃-[6, 19, 19]-²H₃ precursor ion with m/z ofabout 559.4. FIG. 15D shows an exemplary product ion spectra (coveringthe m/z range of about 250 to 350) for fragmentation of the1,25-dihydroxyvitamin D₃-[6, 19, 19]-²H₃—PTAD precursor ion with m/z ofabout 577.4. Details are described in Example 15.

FIG. 16A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 520 to 620) for PTAD-1α,25-dihydroxyvitamin D₃-[26, 26, 26, 27,27, 27]-²H₆ ions. FIG. 16B shows an exemplary product ion spectra(covering the m/z range of about 250 to 350) for fragmentation of thePTAD-1α,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ precursorion with m/z of about 544.4. FIG. 16C shows an exemplary product ionspectra (covering the m/z range of about 250 to 350) for fragmentationof the PTAD-1α,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆precursor ion with m/z of about 562.4. FIG. 16D shows an exemplaryproduct ion spectra (covering the m/z range of about 250 to 350) forfragmentation of the PTAD-1α,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27,27]-²H₆ precursor ion with m/z of about 580.4. Details are described inExample 15.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for measuring vitamin D metabolites in a sample.More specifically, mass spectrometric methods are described fordetecting and quantifying vitamin D metabolites in a sample. The methodsutilize Cookson-type reagents, such as PTAD, to generate derivatizedvitamin D metabolites, and may use an extraction chromatographytechnique, such as turbulent flow liquid chromatography (TFLC), toperform a purification of the derivatized analytes, combined withmethods of mass spectrometry (MS), thereby providing a high-throughputassay system for detecting and quantifying vitamin D metabolites in asample. In some methods, no chromatography, including extractionchromatography, is necessary for sample analysis. In these methods, thederivatized vitamin D metabolites are ionized with LDTD. Preferredembodiments are particularly well suited for application in largeclinical laboratories for automated vitamin D metabolite quantification.

Suitable test samples for use in methods of the present inventioninclude any test sample that may contain the analyte of interest. Insome preferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Preferred samples comprise bodilyfluids such as blood, plasma, serum, saliva, cerebrospinal fluid, ortissue samples; preferably plasma (including EDTA and heparin plasma)and serum; most preferably serum. Such samples may be obtained, forexample, from a patient; that is, a living person, male or female,presenting oneself in a clinical setting for diagnosis, prognosis, ortreatment of a disease or condition.

The present invention also contemplates kits for a vitamin D metabolitequantitation assay. A kit for a vitamin D metabolite quantitation assaymay include a kit comprising the compositions provided herein. Forexample, a kit may include packaging material and measured amounts of aCookson-type reagent and an isotopically labeled internal standard, inamounts sufficient for at least one assay. Typically, the kits will alsoinclude instructions recorded in a tangible form (e.g., contained onpaper or an electronic medium) for using the packaged reagents for usein a vitamin D metabolite quantitation assay.

Calibration and QC pools for use in embodiments of the present inventionare preferably prepared using a matrix similar to the intended samplematrix. If no appropriate biological matrix is commercially availablewith an endogenous concentration of 25OHD₃ sufficiently low to act as ablank, a solution of 5% bovine serum albumin in phosphate bufferedsaline (with an estimated 1.5 ng/mL 25OHD₃) may be used for calibrationand QC pools.

Sample Preparation for Mass Spectrometric Analysis

In preparation for mass spectrometric analysis, vitamin D metabolitesmay be enriched relative to one or more other components in the sample(e.g. protein) by various methods known in the art, including forexample, liquid chromatography, filtration, centrifugation, thin layerchromatography (TLC), electrophoresis including capillaryelectrophoresis, affinity separations including immunoaffinityseparations, extraction methods including ethyl acetate or methanolextraction, and the use of chaotropic agents or any combination of theabove or the like.

Protein precipitation is one method of preparing a test sample,especially a biological test sample, such as serum or plasma. Proteinpurification methods are well known in the art, for example, Polson etal., Journal of Chromatography B 2003, 785:263-275, describes proteinprecipitation techniques suitable for use in methods of the presentinvention. Protein precipitation may be used to remove most of theprotein from the sample leaving vitamin D metabolites in thesupernatant. The samples may be centrifuged to separate the liquidsupernatant from the precipitated proteins; alternatively the samplesmay be filtered to remove precipitated proteins. The resultantsupernatant or filtrate may then be applied directly to massspectrometry analysis; or alternatively to liquid chromatography andsubsequent mass spectrometry analysis. In certain embodiments, samples,such as plasma or serum, may be purified by a hybrid proteinprecipitation/liquid-liquid extraction method. In these embodiments, asample is mixed with methanol, ethyl acetate, and water, and theresulting mixture is vortexed and centrifuged. The resulting supernatantis removed, dried to completion and reconstituted in acetonitrile. Thepurified vitamin D metabolites may then be derivatized with anyCookson-type reagent, preferably PTAD or an isotopically labeled variantthereof.

Another method of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Certain methods of liquidchromatography, including HPLC, rely on relatively slow, laminar flowtechnology. Traditional HPLC analysis relies on column packing in whichlaminar flow of the sample through the column is the basis forseparation of the analyte of interest from the sample. The skilledartisan will understand that separation in such columns is a diffusionalprocess and may select LC, including HPLC, instruments and columns thatare suitable for use with derivatized vitamin D metabolites. Thechromatographic column typically includes a medium (i.e., a packingmaterial) to facilitate separation of chemical moieties (i.e.,fractionation). The medium may include minute particles, or may includea monolithic material with porous channels. A surface of the mediumtypically includes a bonded surface that interacts with the variouschemical moieties to facilitate separation of the chemical moieties. Onesuitable bonded surface is a hydrophobic bonded surface such as an alkylbonded, cyano bonded surface, or highly pure silica surface. Alkylbonded surfaces may include C-4, C-8, C-12, or C-18 bonded alkyl groups.In preferred embodiments, the column is a highly pure silica column(such as a Thermo Hypersil Gold Aq column). The chromatographic columnincludes an inlet port for receiving a sample and an outlet port fordischarging an effluent that includes the fractionated sample. Thesample may be supplied to the inlet port directly, or from an extractioncolumn, such as an on-line SPE cartridge or a TFLC extraction column.

In one embodiment, the sample may be applied to the LC column at theinlet port, eluted with a solvent or solvent mixture, and discharged atthe outlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytyptic(i.e. mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, HPLC is conducted with an alkyl bondedanalytical column chromatographic system. In certain preferredembodiments, a highly pure silica column (such as a Thermo Hypersil GoldAq column) is used. In certain preferred embodiments, HPLC and/or TFLCare performed using HPLC Grade water as mobile phase A and HPLC Gradeethanol as mobile phase B.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, an extraction column may be used for purificationof vitamin D metabolites prior to mass spectrometry. In suchembodiments, samples may be extracted using a extraction column whichcaptures the analyte, then eluted and chromatographed on a secondextraction column or on an analytical HPLC column prior to ionization.For example, sample extraction with a TFLC extraction column may beaccomplished with a large particle size (50 μm) packed column. Sampleeluted off of this column may then be transferred to an HPLC analyticalcolumn for further purification prior to mass spectrometry. Because thesteps involved in these chromatography procedures may be linked in anautomated fashion, the requirement for operator involvement during thepurification of the analyte can be minimized. This feature may result insavings of time and costs, and eliminate the opportunity for operatorerror.

In some embodiments, protein precipitation is accomplished with a hybridprotein precipitation/liquid-liquid extraction method which includesmethanol protein precipitation and ethyl acetate/water extraction fromserum. The resulting vitamin D metabolites may be derivatized prior tobeing subjected to an extraction column. Preferably, the hybrid proteinprecipitation/liquid-liquid extraction method and the extraction columnare connected in an online fashion. In preferred embodiments, theextraction column is a C-8 extraction column, such as a CohesiveTechnologies C8XL online extraction column (50 μm particle size, 0.5×50mm) or equivalent. The eluent from the extraction column may then beapplied to an analytical LC column, such as a HPLC column in an on-linefashion, prior to mass spectrometric analysis. Because the stepsinvolved in these chromatography procedures may be linked in anautomated fashion, the requirement for operator involvement during thepurification of the analyte can be minimized. This feature may result insavings of time and costs, and eliminate the opportunity for operatorerror.

Detection and Quantitation by Mass Spectrometry

In various embodiments, derivatized vitamin D metabolites may be ionizedby any method known to the skilled artisan. Mass spectrometry isperformed using a mass spectrometer, which includes an ion source forionizing the fractionated sample and creating charged molecules forfurther analysis. For example ionization of the sample may be performedby electron ionization, chemical ionization, electrospray ionization(ESI), photon ionization, atmospheric pressure chemical ionization(APCI), photoionization, atmospheric pressure photoionization (APPI),Laser diode thermal desorption (LDTD), fast atom bombardment (FAB),liquid secondary ionization (LSI), matrix assisted laser desorptionionization (MALDI), field ionization, field desorption,thermospray/plasmaspray ionization, surface enhanced laser desorptionionization (SELDI), inductively coupled plasma (ICP) and particle beamionization. The skilled artisan will understand that the choice ofionization method may be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

Derivatized vitamin D metabolites may be ionized in positive or negativemode. In preferred embodiments, derivatized vitamin D metabolites areionized by APCI or LDTD in positive ion mode.

In mass spectrometry techniques generally, after the sample has beenionized, the positively or negatively charged ions thereby created maybe analyzed to determine a mass-to-charge ratio. Suitable analyzers fordetermining mass-to-charge ratios include quadrupole analyzers, iontraps analyzers, and time-of-flight analyzers. Exemplary ion trapmethods are described in Bartolucci, et al., Rapid Commun. MassSpectrom. 2000, 14:967-73.

The ions may be detected using several detection modes. For example,selected ions may be detected, i.e. using a selective ion monitoringmode (SIM), or alternatively, mass transitions resulting from collisioninduced dissociation or neutral loss may be monitored, e.g., multiplereaction monitoring (MRM) or selected reaction monitoring (SRM).Preferably, the mass-to-charge ratio is determined using a quadrupoleanalyzer. For example, in a “quadrupole” or “quadrupole ion trap”instrument, ions in an oscillating radio frequency field experience aforce proportional to the DC potential applied between electrodes, theamplitude of the RF signal, and the mass/charge ratio. The voltage andamplitude may be selected so that only ions having a particularmass/charge ratio travel the length of the quadrupole, while all otherions are deflected. Thus, quadrupole instruments may act as both a “massfilter” and as a “mass detector” for the ions injected into theinstrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

Alternate modes of operating a tandem mass spectrometric instrumentinclude product ion scanning and precursor ion scanning. For adescription of these modes of operation, see, e.g., E. Michael Thurman,et al., Chromatographic-Mass Spectrometric Food Analysis for TraceDetermination of Pesticide Residues, Chapter 8 (Amadeo R.Fernandez-Alba, ed., Elsevier 2005) (387).

The results of an analyte assay may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, external standards may be run with thesamples, and a standard curve constructed based on ions generated fromthose standards. Using such a standard curve, the relative abundance ofa given ion may be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of vitamin Dmetabolites. Methods of generating and using such standard curves arewell known in the art and one of ordinary skill is capable of selectingan appropriate internal standard. For example, in preferred embodimentsone or more isotopically labeled vitamin D metabolites (e.g., 25OHD₂-[6,19, 19]-²H₃ and 25OHD₃-[6, 19, 19]-²H₃) may be used as internalstandards. Numerous other methods for relating the amount of an ion tothe amount of the original molecule will be well known to those ofordinary skill in the art.

As used herein, an “isotopic label” produces a mass shift in the labeledmolecule relative to the unlabeled molecule when analyzed by massspectrometric techniques. Examples of suitable labels include deuterium(²H), ¹³C, and ¹⁵N. For example, 25OHD₂-[6, 19, 19]-²H₃ has a mass aboutthree units higher than 25OHD₂. The isotopic label can be incorporatedat one or more positions in the molecule and one or more kinds ofisotopic labels can be used on the same isotopically labeled molecule.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activated dissociation (CAD) isoften used to generate fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In particularly preferred embodiments, vitamin D metabolites in a sampleare detected and/or quantified using MS/MS as follows. The samples arefirst purified by protein precipitation or a hybrid proteinprecipitation/liquid-liquid extraction. Then, one or more vitamin Dmetabolites in the purified sample are derivatized with a Cookson-typereagent, such as PTAD. The purified samples are then subjected to liquidchromatography, preferably on an extraction column (such as a TFLCcolumn) followed by an analytical column (such as a HPLC column); theflow of liquid solvent from a chromatographic column enters thenebulizer interface of an MS/MS analyzer; and the solvent/analytemixture is converted to vapor in the heated charged tubing of theinterface. The analyte(s) (e.g., derivatized vitamin D metabolites),contained in the solvent, are ionized by applying a large voltage to thesolvent/analyte mixture. As the analytes exit the charged tubing of theinterface, the solvent/analyte mixture nebulizes and the solventevaporates, leaving analyte ions. The ions, e.g. precursor ions, passthrough the orifice of the instrument and enter the first quadrupole.Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowing selection ofions (i.e., selection of “precursor” and “fragment” ions in Q1 and Q3,respectively) based on their mass to charge ratio (m/z). Quadrupole 2(Q2) is the collision cell, where ions are fragmented. The firstquadrupole of the mass spectrometer (Q1) selects for molecules with themass to charge ratios of derivatized vitamin D metabolites of interest.Precursor ions with the correct mass/charge ratios are allowed to passinto the collision chamber (Q2), while unwanted ions with any othermass/charge ratio collide with the sides of the quadrupole and areeliminated. Precursor ions entering Q2 collide with neutral argon gasmolecules and fragment. The fragment ions generated are passed intoquadrupole 3 (Q3), where the fragment ions of derivatized vitamin Dmetabolites of interest are selected while other ions are eliminated.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably positive ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of derivatized vitaminD metabolites that may be used for selection in quadrupole 3 (Q3).

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC-MS methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, may be measured andcorrelated to the amount of the analyte of interest. In certainembodiments, the area under the curves, or amplitude of the peaks, forfragment ion(s) and/or precursor ions are measured to determine theamount of a particular vitamin D metabolite. As described above, therelative abundance of a given ion may be converted into an absoluteamount of the original analyte using calibration standard curves basedon peaks of one or more ions of an internal molecular standard.

EXAMPLES Example 1 Hybrid Protein Precipitation/Liquid-Liquid Extractionand Cookson-Type Derivatization

The following automated hybrid protein precipitation/liquid-liquidextraction technique was conducted on patient serum samples. Gel BarrierSerum (i.e., serum collected in Serum Separator Tubes) as well as EDTAplasma and Heparin Plasma have also been established as acceptable forthis assay.

A Perkin-Elmer Janus robot and a TomTec Quadra Tower robot was used toautomate the following procedure. For each sample, 50 μL of serum wasadded to a well of a 96 well plate. Then 25 μL of internal standardcocktail (containing isotopically labeled 25OHD₂-[6, 19, 19]-²H₃ and25OHD₃-[6, 19, 19]-²H₃) was added to each well, and the plate vortexed.Then 75 μL of methanol was added, followed by additional vortexing. 300μL of ethyl acetate and 75 μL of water was then added, followed byadditional vortexing, centrifugation, and transfer of the resultingsupernatant to a new 96-well plate.

The transferred liquid in the second 96-well plate from Example 1 wasdried to completion under a flowing nitrogen gas maniforld.Derivatization was accomplished by adding 100 μL of a 0.1 mg/mL solutionof the Cookson-type derivatization agent PTAD in acetonitrile to eachwell. The derivatization reaction was allowed to proceed forapproximately one hour, and was quenched by adding 100 μL of water tothe reaction mixture.

Example 2 Extraction of Vitamin D Metabolites with Liquid Chromatography

Sample injection was performed with a Cohesive Technologies Aria TX-4TFLC system using Aria OS V 1.5.1 or newer software.

The TFLC system automatically injected an aliquot of the above preparedsamples into a Cohesive Technologies C8XL online extraction column (50μm particle size, 005×50 mm, from Cohesive Technologies, Inc.) packedwith large particles. The samples were loaded at a high flow rate tocreate turbulence inside the extraction column. This turbulence ensuredoptimized binding of derivatized vitamin D metabolites to the largeparticles in the column and the passage of excess derivatizing reagentand debris to waste.

Following loading, the sample was eluted off to the analytical column, aThermo Hypersil Gold Aq analytical column (5 μm particle size, 50×2.1mm), with a water/ethanol elution gradient. The HPLC gradient wasapplied to the analytical column, to separate vitamin D metabolites fromother analytes contained in the sample. Mobile phase A was water andmobile phase B was ethanol. The HPLC gradient started with a 35% organicgradient which was ramped to 99% in approximately 65 seconds.

Example 3 Detection and Quantitation of Derivatized Vitamin DMetabolites by MS/MS

MS/MS was performed using a Finnigan TSQ Quantum Ultra MS/MS system(Thermo Electron Corporation). The following software programs, all fromThermo Electron, were used in the Examples described herein: QuantumTune Master V 1.5 or newer, Xcalibur V 2.07 or newer, LCQuan V 2.56(Thermo Finnigan) or newer, and ARIA OS v1.5.1 (Cohesive Technologies)or newer. Liquid solvent/analyte exiting the analytical column flowed tothe nebulizer interface of the MS/MS analyzer. The solvent/analytemixture was converted to vapor in the tubing of the interface. Analytesin the nebulized solvent were ionized by ESI.

Ions passed to the first quadrupole (Q1), which selected ions for aderivatized vitamin D metabolite. Ions with a m/z of 570.32±0.50 wereselected for PTAD-25OHD₂; ions with a m/z of 558.32±0.50 were selectedfor PTAD-25OHD₃. Ions entering quadrupole 2 (Q2) collided with argon gasto generate ion fragments, which were passed to quadrupole 3 (Q3) forfurther selection. Mass spectrometer settings are shown in Table 1.Simultaneously, the same process using isotope dilution massspectrometry was carried out with internal standards, PTAD-25OHD₂-[6,19, 19]-²H₃ and PTAD-25OHD₃-[6, 19, 19]-²H₃. The following masstransitions were used for detection and quantitation during validationon positive polarity.

TABLE 1 Mass Spectrometer Settings for Detection of PTAD-25OHD₂ andPTAD-25OHD₃ Mass Spectrometric Instrument Settings Discharge Current 4.0μA Vaporizer Temperature 300 C. Sheath Gas Pressure 15 Ion Sweep GasPressure 0.0 Aux Gas Pressure 5 Capillary Temperature 300 C. SkimmerOffset −10 V Collision Pressure 1.5 mTorr Collision Cell Energy   15 V

TABLE 2 Mass Transitions for PTAD-25OHD₂, PTAD- 25OHD₂-[6, 19, 19]-²H₃(IS), PTAD-25OHD₃, and PTAD-25OHD₃-[6, 19, 19]-²H₃ (IS) (PositivePolarity) Precursor Ion Product Ion Analyte (m/z) (m/z) PTAD-25OHD₂570.32 298.09 PTAD-25OHD₂-[6, 19, 19]-²H₃ (IS) 573.32 301.09 PTAD-25OHD₃558.32 298.09 PTAD-25OHD₃-[6, 19, 19]-²H₃ (IS) 561.32 301.09

Exemplary chromatograms for PTAD-25OHD₃, PTAD-25OHD₃-[6, 19, 19]-²H₃(internal standard), PTAD-25OHD₂, and PTAD-25OHD₂-[6, 19, 19]-²H₃(internal standard), are shown in FIGS. 1A, 1B, 1C, and 1D,respectively.

Exemplary calibration curves for the determination of 25OHD₂ and 25OHD₃in serum specimens are shown in FIGS. 2A and 2B, respectively.

Example 4 Analytical Sensitivity: Lower Limit of Quantitation (LLOQ) andLimit of Detection (LOD)

The LLOQ is the point where measurements become quantitativelymeaningful. The analyte response at this LLOQ is identifiable, discreteand reproducible with a precision (i.e., coefficient of variation (CV))of greater than 20% and an accuracy of 80% to 120%. The LLOQ wasdetermined by assaying five different human serum samples spiked withPTAD-25OHD₂ and PTAD-25OHD₃ at levels near the expected LLOQ andevaluating the reproducibility. Analysis of the collected data indicatesthat samples with concentrations of about 4 ng/mL yielded CVs of about20%. Thus, the LLOQ of this assay for both PTAD-25OHD₂ and PTAD-25OHD₃was determined to be 4 ng/mL. The graphical representations of CV versusconcentration for both analytes are shown in FIGS. 3A-B (FIG. 3A showsthe plots over an expanded concentration range, while FIG. 3B shows thesame plot expanded near the LOQ).

The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as three standarddeviations from the zero concentration. To determine the LOD, generally,blank samples of the appropriate matrix are obtained and tested forinterferences. However, no appropriate biological matrix could beobtained where the endogenous concentration of 25OHD₃ is zero, so asolution of 5% bovine serum albumin in phosphate buffered saline (withan estimated 1.5 ng/mL 25OHD₃) was used for LOD studies. The standardwas run in 20 replicates each and the resulting area rations werestatistically analyzed to determine that the LOD for 25OHD₂ and 25OHD₃are 1.9 and 3.3 ng/mL, respectively. Raw data from these studies ispresented in Table 3, below

TABLE 3 Limit of Detection Raw Data and Analysis Replicate 25OHD₂(ng/mL) 25OHD₃ (ng/mL) 1 0.0 0.0 2 1.1 2.0 3 0.1 2.4 4 0.3 1.1 5 0.5 1.96 0.4 1.8 7 0.2 1.9 8 0.5 2.3 9 1.1 2.3 10 0.5 2.1 11 0.4 1.5 12 1.2 1.913 0.4 1.8 14 0.3 1.6 15 0.0 1.3 16 0.9 1.3 17 0.8 1.5 18 0.1 1.9 19 0.51.7 20 0.4 1.8 Mean 0.4 1.7 SD 0.5 0.5 LOD (Mean + 3SD) 1.9 3.3

Example 5 Reportable Range and Linearity

Linearity of derivatized vitamin D metabolite detection in the assay wasdetermined by diluting four pools serum with high endogenousconcentration of either 25OHD₂ or 25OHD₃ and analyzing undilutedspecimens and diluted specimens at 1:2, 1:4, and 1:8, in quadruplicate.Quadratic regression of the data was performed yielding correlationcoefficients across the concentration range tested of R²=0.97. Thesestudies demonstrated that specimens may be diluted at 1:4 with averagerecovery of 101%, permitting a reportable range of about 4 to about 512ng/mL. Average measured values for each of the specimen dilution levelsand correlation values from linear regression analysis are presented inTable 4A, below. Percent recoveries for each of the specimen dilutionlevels are presented in Table 4B, below.

TABLE 4A Linearity Data and Linear Regression Analysis over ReportableRange 25OHD₂ (ng/mL) 25OHD₃ (ng/mL) Dilution Level Pool 1 Pool 2 Pool 1Pool 2 Undiluted 110.0 75.6 73.3 60.6 1:2 55.5 39.3 35.7 28.7 1:4 26.219.4 18.1 16.3 1:8 14.3 10.9 9.7 8.3 R² 0.9744 0.9721 0.9705 0.9601

TABLE 4B Percent Recovery at Various Specimen Dilution Levels 25OHD₂(ng/mL) 25OHD₃ (ng/mL) Dilution Level Pool 1 Pool 2 Pool 1 Pool 2Undiluted (100%) (100%) (100%) (100%) 1:2 100.9 104 97.4 94.8 1:4 95.5102.7 98.6 107.3 1:8 104.2 115.0 106.0 109.0

Example 6 Analyte Specificity

The specificity of the assay against similar analytes was determined tohave no cross reactivity for any vitamin D metabolite tested with theexception of 3-epi-25OHD₃, which behaves similarly to 25OHD₃ in theassay. The side-chain labeled stable isotopes of 25OHD2 and 25OHD₃ alsoshowed cross-reactivity owning to hydrogen exchange that occurs in theion source. Thus, side-chain labeled stable isotopes of 25OHD₂ and25OHD₃ should not be used as internal standards. Table 5, below, showsthe compounds tested and the results of the cross-reactivity studies.

TABLE 5 Cross-Reactivity Studies (Compounds tested and results) Cross-Reac- Analyte 25OHD₂ 25OHD₃ tivity 1,25(OH)₂D₃ — — No 1,25(OH)₂D₂ — — No1,25(OH)₂D₃-[6,19,19′]-²H — — No 1,25(OH)₂D₃-[26,26,26,27,27,27]-²H — —No 1,25(OH)₂D₂-[26,26,26,27,27,27]-²H — — No 25OHD₃ —  (100%) — 25OHD₂(100%) — — 25OHD₃-IS-[6,19,19′]-²H — — No 25OHD₂-IS-[6,19,19′]-²H — — No25OHD₃-IS-[26,26,26,27,27,27]-²H — 13.8% Yes25OHD₂-IS-[26,26,26,27,27,27]-²H  2.7% — Yes vitamin D₃ — — No vitaminD₂ — — No vitamin D₃-[6,19,19′]-²H — — No vitamin D₂-[6,19,19′]-²H — —No vitamin D₃-[26,26,26,27,27,27]-²H — — No vitaminD₂-[26,26,26,27,27,27]-²H — — No 1-OH-D₃ (Alfacalcidiol) — — No 1-OH-D₂(Hectoral) — — No 24,25(OH)₂D₃ — — No 25,26(OH)₂D₃ — — No 3-epi-25OHD₃ —— No 3-epi-1,25(OH)₂D₃ — 33.3% Yes Dihydrotachysterol — — No1,25(OH)₂D₃-26,23-lactone — — No Paracalcitol (Zemplar) — — NoCalcipotriene (Dovonex) — — No 7-Dehydrocholesterol — — No

Example 7 Reproducibility

Six standards at 5, 15, 30, 60, 90, and 120 ng/mL for each analyte wererun in every assay as a means as quantitating reproducibility. Theday-to-day reproducibility was determined using calibration curves from19 assays. The data from these 19 assays are presented in Tables 6A (for25OHD₂) and 6B (for 25OHD₃).

TABLE 6A Standard curves demonstrate reproducibility of PTAD-25OHD₂determination Concentration 5 15 30 60 90 120 Assay ng/mL ng/mL ng/mLng/mL ng/mL ng/mL  1 0.06 0.16 0.36 0.68 0.92 1.23  2 0.08 0.17 0.360.61 0.94 1.18  3 0.07 0.17 0.32 0.66 0.92 1.19  4 0.06 0.19 0.29 0.690.98 1.16  5 0.07 0.15 0.37 0.60 0.85 1.13  6 0.07 0.16 0.32 0.64 0.951.20  7 0.07 0.16 0.35 0.63 0.99 1.18  8 0.06 0.16 0.35 0.60 0.98 1.31 9 0.06 0.18 0.32 0.66 0.96 1.10 10 0.06 0.15 0.35 0.62 0.89 1.22 110.05 0.17 0.33 0.65 0.96 1.17 12 0.04 0.17 0.32 0.61 0.97 1.12 13 0.050.16 0.34 0.62 0.97 1.30 14 0.06 0.17 0.31 0.61 0.95 1.21 15 0.07 0.160.34 0.70 0.94 1.30 16 0.08 0.17 0.39 0.70 1.06 1.27 17 0.06 0.15 0.360.65 1.03 1.20 18 0.05 0.18 0.34 0.81 0.91 1.33 19 0.06 0.17 0.30 0.621.06 1.21 Avg 0.06 0.16 0.34 0.65 0.96 1.21 SD 0.01 0.01 0.02 0.05 0.050.07 CV % 15.4 6.3 7.4 8.0 5.6 5.4

TABLE 6B Standard curves demonstrate reproducibility of PTAD-25OHD₃determination Concentration 5 15 30 60 90 120 Assay ng/mL ng/mL ng/mLng/mL ng/mL ng/mL  1 0.07 0.16 0.36 0.61 0.95 1.19  2 0.07 0.17 0.320.66 1.01 1.12  3 0.06 0.16 0.32 0.60 1.00 1.16  4 0.06 0.17 0.31 0.600.94 1.09  5 0.05 0.16 0.33 0.65 0.96 1.11  6 0.07 0.17 0.34 0.65 0.871.13  7 0.07 0.17 0.31 0.61 0.95 1.21  8 0.06 0.15 0.29 0.58 0.90 1.21 9 0.07 0.17 0.32 0.65 0.88 1.15 10 0.06 0.14 0.30 0.57 1.05 1.16 110.06 0.15 0.30 0.56 0.87 1.15 12 0.05 0.15 0.31 0.64 0.85 1.06 13 0.060.16 0.33 0.60 0.88 1.08 14 0.06 0.17 0.31 0.61 0.91 1.22 15 0.06 0.180.34 0.66 0.96 1.18 16 0.06 0.17 0.35 0.65 0.94 1.21 17 0.06 0.17 0.360.64 0.94 1.17 18 0.07 0.17 0.34 0.66 0.98 1.18 19 0.07 0.16 0.34 0.680.84 1.27 Avg 0.06 0.16 0.33 0.63 0.93 1.16 SD 0.00 0.01 0.02 0.03 0.060.05 CV % 7.9 5.8 5.9 5.5 6.1 4.6

Example 8 Intra-Assay and Inter-Assay Variation Studies

Intra-assay variation is defined as the reproducibility of results for asample within a single assay. To assess intra-assay variation, twentyreplicates from each of four quality control (QC) pools covering thereportable range of the assay were prepared and measured from pooledserum with 25OHD₂ and 25OHD₃ at arbitrary ultralow, low, medium, andhigh concentrations for each analyte. Acceptable levels for thecoefficient of variation (CV) are less then 15% for the three higherconcentration, and less than 20% for the lowest concentration (at ornear the LOQ for the assay).

The results of the intra-assay variation studies indicate that the CVfor the four QC pools are 9.1%, 6.4%, 5.0%, and 5.9% with meanconcentrations of 13.7 ng/mL, 30.0 ng/mL, 52.4 ng/mL, and 106.9 ng/mL,respectively, for PTAD-25OHD₂, and 3.5%, 4.9%, 5.1%, and 3.3% with meanconcentrations of 32.8 ng/mL, 15.0 ng/mL, 75.4 ng/mL, and 102.3 ng/mL,respectively, for PTAD-25OHD₃. The data from analysis of thesereplicates is shown in Tables 7A and 7B.

TABLE 7A PTAD-25OHD₂ Intra-assay variation studies QC (U) QC (L) QC (M)QC (H) Lot # Lot # Lot # Lot # Repli- 090837 090838 090839 090840 cateng/mL ng/mL ng/mL ng/mL 1 15.2 31.4 49.5 108.9 2 12.3 29.7 53.2 109.3 313.8 30.8 50.9 98.9 4 12.4 30.1 50.4 111.5 5 14.6 27.2 49.7 109.0 6 14.629.1 47.6 110.3 7 13.6 33.0 53.3 95.6 8 11.4 29.9 53.3 98.5 9 14.0 31.555.2 110.7 10 13.7 29.1 49.0 113.5 11 13.7 29.5 56.8 100.4 12 13.0 25.554.1 105.4 13 15.6 34.2 53.6 102.0 14 11.7 28.7 52.9 103.2 15 13.5 28.149.4 121.0 16 13.6 29.8 52.0 102.9 17 13.1 29.4 56.8 113.4 18 14.4 30.654.5 103.3 19 16.2 31.6 53.1 110.8 20 12.7 30.7 — 110.4 Avg 0.06 0.160.33 0.63 SD 0.00 0.01 0.02 0.03 CV % 7.9 5.8 5.9 5.5

TABLE 7B PTAD-25OHD₃ Intra-assay variation studies QC (U) QC (L) QC (M)QC (H) Lot # Lot # Lot # Lot # Repli- 090837 090838 090839 090840 cateng/mL ng/mL ng/mL ng/mL 1 34.4 13.7 75.7 101.7 2 35.0 14.2 78.7 101.8 333.2 14.7 73.1 103.2 4 34.4 14.9 83.7 104.1 5 32.4 14.5 72.7 107.0 633.3 14.3 73.6 107.6 7 33.8 15.0 79.1 97.5 8 32.1 15.8 73.1 98.7 9 32.415.5 74.2 106.5 10 31.4 15.4 74.5 106.1 11 31.8 14.7 69.3 105.9 12 31.216.8 73.5 97.7 13 34.1 15.4 72.7 104.9 14 33.8 15.3 75.1 99.8 15 32.015.7 76.2 102.2 16 33.2 14.7 74.2 102.2 17 32.6 14.7 85.0 100.5 18 31.613.9 75.5 101.8 19 31.3 15.6 73.6 99.9 20 32.5 15.3 — 96.3 Avg 32.8 15.075.4 102.3 SD 1.1 0.7 3.8 3.4 CV % 3.5 4.9 5.1 3.3

Five aliquots of each of the same four QC pools were assayed over sixdays to determine the coefficient of variation (CV) between assays. Theresults of the intra-assay variation studies indicate that theinter-assay CV for the four QC pools are 8.3%, 6.2%, 8.1%, and 6.4% withmean concentrations of 13.1 ng/mL, 29.8 ng/mL, 51.9 ng/mL, and 107.8ng/mL, respectively, for PTAD-25OHD₂, and 4.8%, 6.7%, 4.7%, and 6.7%with mean concentrations of 31.1 ng/mL, 14.5 ng/mL, 75.1 ng/mL, and108.4 ng/mL, respectively, for PTAD-25OHD₃. The data from analysis ofthese replicates is shown in Tables 8A and 8B.

TABLE 8A PTAD-25OHD₂ Inter-assay variation studies QC (U) QC (L) QC (M)QC (H) Lot # Lot # Lot # Lot # 090837 090838 090839 090840 Assay ng/mLng/mL ng/mL ng/mL 1 13.6 28.1 51.7 119.6 12.8 30.1 49.4 117.9 14.6 32.049.7 105.1 13.0 30.8 52.3 100.2 13.0 29.2 56.6 110.3 2 12.9 31.3 46.3108.1 13.5 30.3 52.1 117.8 10.9 29.7 46.9 105.8 11.2 30.6 43.6 105.212.8 28.7 50.3 104.9 3 12.6 28.8 56.5 115.3 16.4 29.3 63.8 103.0 13.226.2 45.5 103.2 11.5 30.8 53.8 113.2 12.4 33.7 51.6 106.9 4 12.1 28.558.5 97.0 13.9 26.2 51.8 115.1 14.4 29.6 48.9 112.2 13.1 32.1 52.3 97.912.6 30.5 52.2 104.2 5 12.7 29.9 54.5 101.3 14.3 28.3 46.3 102.2 13.930.0 56.1 111.4 13.1 32.6 51.2 123.1 12.4 26.2 51.2 98.3 6 12.5 30.650.1 104.6 12.9 32.6 51.8 104.8 14.0 28.6 53.7 108.9 14.3 29.1 51.0113.8 12.9 29.1 56.4 102.2 Avg 13.1 29.8 51.9 107.8 SD 1.1 1.8 4.2 6.8CV % 8.3 6.2 8.1 6.4

TABLE 8B PTAD-25OHD₃ Inter-assay variation studies QC (U) QC (L) QC (M)QC (H) Lot # Lot # Lot # Lot # 090837 090838 090839 090840 Assay ng/mLng/mL ng/mL ng/mL 1 32.6 13.4 76.7 104.9 30.0 12.7 77.6 107.0 34.1 15.478.4 107.1 34.0 14.8 76.6 105.1 30.2 15.5 74.8 110.2 2 33.5 13.2 69.8109.8 32.4 14.3 75.0 106.4 30.2 16.2 73.4 112.1 31.4 16.1 71.9 97.0 31.413.7 75.2 117.5 3 31.5 13.3 70.2 112.4 32.1 14.6 82.6 101.5 31.0 15.470.8 99.8 28.7 15.6 74.3 103.6 30.7 15.1 79.8 99.1 4 31.9 14.5 76.3124.2 27.5 14.0 70.5 113.6 27.9 14.8 74.5 112.5 32.1 16.1 74.3 108.831.0 14.4 74.5 110.1 5 31.2 13.1 76.7 96.5 31.5 13.5 82.9 106.1 31.514.7 70.9 112.9 30.9 14.5 77.6 117.7 31.0 13.9 73.1 101.9 6 29.8 15.673.3 110.1 30.5 13.5 71.5 99.3 31.0 13.9 72.6 120.5 30.5 14.6 74.2 109.430.7 13.6 81.8 115.9 Avg 31.1 14.5 75.1 108.4 SD 1.5 1.0 3.6 6.9 CV %4.8 6.7 4.7 6.4

Example 9 Recovery Studies

Two recovery studies were performed. The first was performed using sixspecimens, spiked with two different concentrations each of 25OHD₂ and25OHD₃. These spiked specimens were analyzed in quadruplicate. Thespiked concentrations were within the workable range of the assay. Thesix pools yielded an average accuracy of 89% at spiked levels of graterthan 44 ng/mL and 92% at spiked levels of greater than 73 ng/mL. Onlytwo of the 24 experimental recoveries were less than 85%; the remaining22 assays were within the acceptable accuracy range of 85-115%. Theresults of the spiked specimen recovery studies are presented in Table9, below.

TABLE 9 Spiked Specimen Recovery Studies 25OHD₂ 25OHD₃ (% (% Pool SpikeLevel ng/mL Recovery) ng/mL Recovery) 1 — 12.0 — 10.8 — 44 ng/ml 25OHD₂48.0 81.2 10.7 — 73 ng/mL 25OHD₂ 79.0 91.6 10.7 — 44 ng/mL 25OHD₃ 12.7 —51.9 92.9 73 ng/mL 25OHD₃ 11.5 — 76.5 89.9 2 — 11.9 — 10.8 — 44 ng/mL25OHD₂ 48.0 81.4 10.6 — 73 ng/mL 25OHD₂ 75.6 87.1 11.0 — 44 ng/mL 25OHD₃10.0 — 48.8 85.6 73 ng/mL 25OHD₃ 11.6 — 76.4 89.7 3 — 13.6 — 6 — 44ng/mL 25OHD₂ 52.5 87.8 10.9 — 73 ng/mL 25OHD₂ 76.8 86.4 10.5 — 44 ng/mL25OHD₃ 13.2 — 49.6 88.0 73 ng/mL 25OHD₃ 12.3 — 78.0 92.2 4 — 9.0 — 12.7— 44 ng/mL 25OHD₂ 50.3 93.1 13.5 — 73 ng/mL 25OHD₂ 77.6 93.8 13.2 — 44ng/mL 25OHD₃ 10.0 — 52.1 89.0 73 ng/mL 25OHD₃ 9.5 — 83.6 97.0 5 — 21.8 —14.0 — 44 ng/mL 25OHD₂ 68.0 104.2  13.3 — 73 ng/mL 25OHD₂ 91.1 94.8 13.6— 44 ng/mL 25OHD₃ 23.3 — 53.5 89.1 73 ng/mL 25OHD₃ 22.2 — 86.4 99.1 6 —13.8 — 9.3 — 44 ng/mL 25OHD₂ 50.6 83.0 9.2 — 73 ng/mL 25OHD₂ 83.9 95.99.5 — 44 ng/mL 25OHD₃ 13.5 — 48.6 88.6 73 ng/mL 25OHD₃ 13.2 — 76.5 91.9

The second recovery study was performed again using six specimens. Ofthese six specimens, three had high endogenous concentration of 25OHD₂and three had high endogenous concentrations of 25OHD₃. The specimenswere paired and mixed at ratios of 4:1, 1:1, and 1:4, and the resultingmixtures analyzed in quadruplicate. These experiments yielded an averageaccuracy of 98% for 25OHD₂ and 93% for 25OHD₃. All individual resultswere within the acceptable accuracy range of 85-115%. The results of themixed specimen recovery studies are presented in Table 10, below.

TABLE 10 Mixed Specimen Recovery Studies 25OHD₂ 25OHD₃ Specimen MeasuredExpected Recovery Measured Expected Recovery Mixture ng/mL ng/mL (%)ng/mL ng/mL (%) 100% A 45.2 — — 5.5 — — 4:1 A:B 37.1 37.0 100  11.6 13.188 1:1 A:B 26.4 24.6 107  24.4 24.4 100  1:4 A:B 12.6 12.3 102  33.935.7 95 100% B 4.1 — — 43.3 — — 100% C 46.8 — — 8.3 — — 4:1 C:D 38.138.7 98 17.7 18.3 97 1:1 C:D 25.0 26.6 94 32.0 33.4 96 1:4 C:D 14.4 14.4100  46.5 48.4 96 100% D 6.3 — — 58.5 — — 100% E 38.7 — — 7.4 — — 4:1E:F 33.4 34.3 97 15.7 17.5 89 1:1 E:F 27.1 27.7 98 27.8 32.6 85 1:4 E:F18.3 21.0 87 44.0 47.7 92 100% F 16.6 — — 57.8 — — *Measured values areaverages of analysis of four aliquots.

Example 10 Method Correlation Study

The method of detecting vitamin D metabolites followingPTAD-derivatization was compared to a mass spectrometric method in whichthe vitamin D metabolites are not derivatized prior to analysis. Such amethod is described in the published U.S. Patent Application2006/0228808 (Caulfield, et al.). Eight specimens were split andanalyzed according to both methods. The correlation between the twomethods was assessed with linear regression, deming regression, andBland-Altman analysis for complete data sets (including calibrationsamples, QC pools, and unknowns), as well as for unknowns only.

Plots of the linear regression analysis and the Deming regressionanalysis are shown in FIGS. 4A-B (for 25OHD₂) and FIGS. 5A-B (for25OHD₃).

Example 11 Hemolysis, Lipemia, and Icteria Studies

The effect hemolysis, lipemia, and icteria have on the assay was alsoinvestigated.

Hemolysis.

The effect of hemolysis was evaluated by pooling patient samples withknown endogenous concentrations of both 25OHD₂ and 25OHD₃ to create fivedifferent pools with concentrations across the dynamic range of theassay. Then, lysed whole blood was spiked into the pools to generatelightly and moderately hemolyzed samples.

The lightly and moderately hemolyzed samples were analyzed inquadruplicate and the results were compared to levels of samples withoutwhole blood spikes. The resulting comparison indicated a % difference ofless than 15% for both 25OHD₂ and 25OHD₃. Therefore, light to moderatelyhemolyzed specimens are acceptable for analysis.

Lipemia.

The effect of lipemia was evaluated by pooling patient samples withknown endogenous concentrations of both 25OHD₂ and 25OHD₃ to create fivedifferent pools with concentrations across the dynamic range of theassay. Then, powdered lipid extract was added to the pools to generatelightly and grossly lipemic specimens. Specimens were run inquadruplicate and results were compared to the non-lipemic pool resultand the accuracy was calculated. The data shows that determination of25OHD₂ is unaffected by lipemia (all values were within an acceptableaccuracy range of 85-115%), however, 25OHD₃ is affected by lipemia,resulting in determination in lower than expected values. The degree ofvariance increased with the degree of lipemia. Therefore, light but notgrossly lipemic specimens are acceptable.

Icteria.

The effect of icteria was evaluated by pooling patient samples withknown endogenous concentrations of both 25OHD₂ and 25OHD₃ to create fivedifferent pools with concentrations across the dynamic range of theassay. Then, a concentrated solution of Bilirubin was spiked into thepools to generate lightly and grossly icteric specimens. Specimens wererun in quadruplicate and results were compared to the non-icteric poolresult and the accuracy was calculated. The data showed that 25OHD₂ and25OHD₃ are unaffected by icteria (with all values within an acceptableaccuracy range of 85-115%). Therefore, icteric specimens are acceptable.

Example 12 Injector Carryover Studies

Blank matrices were run immediately after a specimen with a highconcentration of 25OHD₂ and 25OHD₃ in order to evaluate carryoverbetween samples. These studies indicated that the response at theretention time of analyte or internal standard was not large enough tocompromise the integrity of the assay. Data from these studies ispresented in Table 11, below.

TABLE 11 Injector Carryover Study Results Specimen 25OHD₂ 25OHD₃Injection Type (ng/mL) (ng/mL) 1 Blank 0.9 1.6 2 High 292.6 356.8 3Blank 1.0 0.9 4 Blank −0.1 0.5 5 High 290.1 360.1 6 High 299.9 350.5 7Blank 1.0 1.5 8 Blank 0.6 1.4 9 Blank 1.3 1.4 10 High 285.8 352.1 11High 303.1 312.1 12 High 293.8 295.1 13 Blank 0.9 0.8 14 Blank 1.0 1.815 Blank 1.1 1.4 16 Blank 1.0 1.6 17 High 291.7 371.6 18 High 334.2360.1 19 High 301.7 328.5 20 High 283.1 382.1 21 Blank 0.6 1.1 22 Blank0.6 1.3 23 Blank 0.7 1.4 24 Blank 0.4 1.9 25 Blank 0.4 0.9 26 High 300.7311.7 27 High 279.5 302.0 28 High 317.5 341.0 29 High 261.5 403.4 30High 288.3 362.6 31 Blank 2.7 1.6 32 Blank 1.7 1.2 33 Blank 0.5 1.3 34Blank 1.3 1.7 35 Blank 0.3 1.6 36 Blank 0.6 1.4 37 High 311.7 366.2 38High 314.1 342.0 39 High 325.7 349.1 40 High 289.6 326.6 41 High 291.5322.3 42 High 278.9 336.5 43 Blank 2.1 2.5 44 Blank 0.6 1.6 45 Blank 0.71.4 46 Blank 0.7 1.5 47 Blank 0.1 1.0 48 Blank 0.7 1.1 49 Blank 1.3 1.050 High 281.2 345.6 51 High 312.5 348.3 52 High 304.8 329.1 53 High290.5 353.9 54 High 286.4 344.9 55 High 302.5 330.6 56 High 292.2 388.557 Blank 0.8 1.5 58 Blank 1.3 1.4 59 Blank 3.5 2.6 60 Blank 0.4 1.8 61Blank 1.0 1.4 62 Blank 1.0 1.2 63 Blank 0.7 1.0 64 Blank 1.1 1.4 65 High285.4 355.4 66 High 318.0 355.0 67 High 285.5 345.7 68 High 303.0 317.169 High 276.3 351.4 70 High 321.8 350.4 71 High 279.4 329.6 72 High299.1 337.9 73 Blank 0.9 1.6 74 Blank 1.7 1.6 75 Blank 1.0 1.1 76 Blank1.8 2.7 77 Blank 1.0 1.9 78 Blank 0.6 1.1 79 Blank 0.9 0.9 80 Blank 1.22.2

Example 13 Suitable Specimen Types

The assay was conducted on various specimen types. Human serum andGel-Barrier Serum (i.e., serum from Serum Separator Tubes), as well asEDTA Plasma and Heparin were established as acceptable sample types. Inthese studies, sets of human serum (serum), Gel-Barrier Serum (SST),EDTA Plasma (EDTA), and heparin (Na Hep) drawn at the same time from thesame patient were tested for 25OHD₂ (40 specimen sets) and 25OHD₃ (6specimen sets). Due to the limitations with clot detection/sensing inexisting automated pipetting systems, plasma was not tested forautomated procedures.

The results of the specimen type studies are presented in Tables 12A andB for 25OHD₂ and 25OHD₃, respectively.

TABLE 12A Results from Specimen Type Studies for 25OHD₂ Specimen CCMeasured Concentration 25OHD₂ (ng/mL) Set ID# Serum SST EDTA Na Hep 25207 6.6 6.9 7.1 7.2 6 5339 5.8 5.2 4.5 5.6 11 5355 7.8 8.2 8.8 8.2 205493 3.9 4.2 4.3 4.2 37 5650 3.7 4.5 4.6 5.2 39 5653 4.7 5.1 4.6 4.7

TABLE 12B Results from Specimen Type Studies for 25OHD₃ Specimen CCMeasured Concentration 25OHD₃ (ng/mL) Set ID# Serum SST EDTA Na Hep 15804 26.8 25.7 24.3 26.8 2 5207 16.1 17.6 16.1 16.5 3 5235 17.4 17.716.8 17.2 4 5333 62.9 62.7 63.7 57.4 5 5336 33.0 32.4 28.8 28.8 6 533917.2 17.6 17.8 17.8 7 5340 16.7 17.1 16.8 16.5 8 5342 28.6 27.9 26.930.5 9 5344 23.3 23.8 22.3 22.9 10 5351 19.4 20.0 20.4 21.4 11 5355 17.616.7 19.4 18.3 12 5362 25.3 25.2 23.5 24.0 13 5365 40.9 44.7 46.8 42.914 5406 23.1 20.3 21.5 20.5 15 5408 31.7 33.9 31.6 32.3 16 5414 21.121.8 21.2 20.4 17 5422 44.0 47.7 45.5 47.3 18 5432 13.6 14.2 12.3 13.819 5463 15.1 15.4 15.6 14.5 20 5493 38.6 42.2 40.1 36.8 21 5366 47.548.1 46.7 45.1 22 5368 23.0 23.6 22.3 22.3 23 5392 34.1 33.4 34.4 27.624 5451 36.4 42.1 40.0 38.3 25 5455 27.3 29.9 25.1 27.9 26 5476 16.717.9 15.8 16.6 27 5483 30.4 28.2 26.5 28.1 28 5484 38.2 37.7 37.2 36.029 5537 30.5 30.3 27.2 27.0 30 5547 9.2 9.0 8.7 8.2 31 5560 9.4 10.9 9.88.6 32 5571 30.9 31.7 29.6 29.2 33 5572 47.6 50.3 47.7 48.6 34 5577 11.211.7 10.4 9.2 35 5606 39.3 38.8 41.0 37.7 36 5611 21.9 25.3 20.7 21.1 375650 38.0 34.3 34.6 36.2 38 5651 34.8 32.8 32.4 32.4 39 5653 29.4 32.328.1 27.0 40 5668 11.4 12.8 14.2 13.1

Example 14 Exemplary Spectra from LDTD-MS/MS Analysis of PTADDerivatized 25-hydroxyvitamin D₂ and PTAD Derivatized 25-hydroxyvitaminD₃

PTAD derivatives of 25-hydroxyvitamin D₂, 25-hydroxyvitamin D₂-[6, 19,19]-²H₃, 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆25-hydroxyvitamin D₃, 25-hydroxyvitamin D₃-[6, 19, 19]-²H₃, and25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ were prepared asdescribed above. The resulting solutions were analyzed by LDTD-MS/MS.Results of these analyses are presented below.

Exemplary Q1 scan spectra from the analysis of samples containingPTAD-25-hydroxyvitamin D₂, PTAD-25-hydroxyvitamin D₂-[6, 19, 19]-²H₃,and PTAD-25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆ are shown inFIGS. 6A, 7A, and 8A, respectively. These spectra were collected byscanning Q1 across a m/z range of about 520 to 620.

Exemplary product ion scans from each of these species are presented inFIGS. 6B, 7B, and 8B, respectively. The precursor ions selected in Q1,and collision energies used in fragmenting the precursors are indicatedin Table 13.

A preferred MRM transition for the quantitation ofPTAD-25-hydroxyvitamin D₂ is fragmenting a precursor ion with a m/z ofabout 570.3 to a product ion with a m/z of about 298.1. A preferred MRMtransition for the quantitation of PTAD-25-hydroxyvitamin D₂-[6, 19,19]-²H₃ is fragmenting a precursor ion with a m/z of about 573.3 to aproduct ion with a m/z of about 301.1. A preferred MRM transition forthe quantitation of PTAD-25-hydroxyvitamin D₂-[26, 26, 26, 27, 27,27]-²H₆ is fragmenting a precursor ion with a m/z of about 576.3 to aproduct ion with a m/z of about 298.1. However, as can be seen in theproduct ion scans in FIGS. 6B, 7B, and 8B, additional product ions maybe selected to replace or augment the preferred fragment ion.

TABLE 13 Precursor Ions and Collision Cell Energies for Fragmentation ofPTAD-25-hydroxyvitamin D₂, PTAD-25-hydroxyvitamin D₂-[6, 19, 19]-²H₃,and PTAD-25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆ CollisionPrecursor Cell Energy Analyte Ion (m/z) (V) PTAD-25-hydroxyvitamin D₂570.3 15 PTAD-25-hydroxyvitamin D₂- 573.3 15 [6,19,19]-²H₃PTAD-25-hydroxyvitamin 576.3 15 D₂-[26,26,26,27,27,27]-²H₆

Exemplary Q1 scan spectra from the analysis of PTAD-25-hydroxyvitaminD₃, PTAD-25-hydroxyvitamin D₃-[6, 19, 19]-²H₃, andPTAD-25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ are shown inFIGS. 9A, 10A, and 11A, respectively. These spectra were collected byscanning Q1 across a m/z range of about 520 to 620.

Exemplary product ion scans from each of these species are presented inFIGS. 9B, 10B, and 11B, respectively. The precursor ions selected in Q1and the collision energies used to generate these product ion spectraare indicated in Table 14.

A preferred MRM transition for the quantitation ofPTAD-25-hydroxyvitamin D₃ is fragmenting a precursor ion with a m/z ofabout 558.3 to a product ion with a m/z of about 298.1. A preferred MRMtransition for the quantitation of PTAD-25-hydroxyvitamin D₃-[6, 19,19]-²H₃ is fragmenting a precursor ion with a m/z of about 561.3 to aproduct ion with a m/z of about 301.1. A preferred MRM transition forthe quantitation of PTAD-25-hydroxyvitamin D₃-[26, 26, 26, 27, 27,27]-²H₆ is fragmenting a precursor ion with a m/z of about 564.3 to aproduct ion with a m/z of about 298.1. However, as can be seen in theproduct ion scans in FIGS. 9B, 10B, and 11B, additional product ions maybe selected to replace or augment the preferred fragment ion.

TABLE 14 Precursor Ions and Collision Cell Energies for Fragmentation ofPTAD-25-hydroxyvitamin D₃, PTAD-25-hydroxyvitamin D₃-[6, 19, 19]-²H₃,and PTAD-25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ CollisionPrecursor Cell Energy Analyte Ion (m/z) (V) PTAD-25-hydroxyvitamin D₃558.3 15 PTAD-25-hydroxyvitamin D₃- 561.3 15 [6,19,19]-²H₃PTAD-25-hydroxyvitamin 564.3 15 D₃-[26,26,26,27,27,27]-²H₆

Example 15 Exemplary Spectra from LDTD-MS/MS Analysis of PTADDerivatized 1α,25-dihydroxyvitamin D₂ and 1α,25-dihydroxyvitamin D₃

PTAD derivatives of 1α,25-dihydroxyvitamin D₂, 1α,25-dihydroxyvitaminD₂-[26, 26, 26, 27, 27, 27]-²H₆, 1α,25-dihydroxyvitamin D₃,1α,25-dihydroxyvitamin D₃-[6, 19, 19]-²H₃₅ and 1α,25-dihydroxyvitaminD₃-[26, 26, 26, 27, 27, 27]-²H₆ were prepared by treating aliquots ofstock solutions of each analyte with PTAD in acetonitrile. Thederivatization reactions was allowed to proceed for approximately onehour, and were quenched by adding water to the reaction mixture. Thederivatized analytes were then analyzed according to the procedureoutlined above in Example 1.

Exemplary Q1 scan spectra from the analysis of samples containingPTAD-1α,25-dihydroxyvitamin D₂ and PTAD-1α,25-dihydroxyvitamin D₂-[26,26, 26, 27, 27, 27]-²H₆ are shown in FIGS. 12A, and 13A, respectively.These spectra were collected by scanning Q1 across a m/z range of about520 to 620.

Exemplary product ion scans generated from three different precursorions for each of PTAD-1α,25-dihydroxyvitamin D₂ andPTAD-1α,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆ arepresented in FIGS. 12B-D, and 13B-D, respectively. The precursor ionsselected in Q1 and the collision energies used to generate these production spectra are indicated in Table 15.

Exemplary MRM transitions for the quantitation ofPTAD-1α,25-dihydroxyvitamin D₂ include fragmenting a precursor ion witha m/z of about 550.4 to a product ion with a m/z of about 277.9;fragmenting a precursor ion with a m/z of about 568.4 to a product ionwith a m/z of about 298.0; and fragmenting a precursor ion with a m/z ofabout 586.4 to a product ion with a m/z of about 314.2. Exemplary MRMtransitions for the quantitation of PTAD-1α,25-dihydroxyvitamin D₂-[26,26, 26, 27, 27, 27]-²H₆ include fragmenting a precursor ion with a m/zof about 556.4 to a product ion with a m/z of about 278.1; fragmenting aprecursor ion with a m/z of about 574.4 to a product ion with a m/z ofabout 298.1; and fragmenting a precursor ion with a m/z of about 592.4to a product ion with a m/z of about 313.9. However, as can be seen inthe product ion scans in FIGS. 12B-D and 13B-D, several other productions are generated upon fragmentation of the precursor ions. Additionalproduct ions may be selected from those indicated in FIGS. 12B-D and13B-D to replace or augment the exemplary fragment ions.

TABLE 15 Precursor Ions and Collision Cell Energies for Fragmentation ofPTAD-1α,25-dihydroxyvitamin D₂ and PTAD-1α,25-dihydroxyvitamin D₂-[26,26, 26, 27, 27, 27]-²H₆ Collision Precursor Cell Energy Analyte Ion(m/z) (V) PTAD-1α,25-dihydroxyvitamin D₂ 550.4, 568.4, 586.4 15PTAD-1α,25-dihydroxyvitamin 556.3, 574.4, 592.4 15D₂-[26,26,26,27,27,27]-²H₆

Exemplary Q1 scan spectra from the analysis of PTAD-1α,25-hydroxyvitaminD₃, PTAD-1α,25-dihydroxyvitamin D₃-[6, 19, 19]-²H₃, andPTAD-1α,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ are shown inFIGS. 14A, 15A, and 16A, respectively. These spectra were collected byscanning Q1 across a m/z range of about 520 to 620.

Exemplary product ion scans generated from three different precursorions for each of PTAD-1α,25-hydroxyvitamin D₃,PTAD-1α,25-dihydroxyvitamin D₃-[6, 19, 19]-²H₃, andPTAD-1α,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ arepresented in FIGS. 14B-D, 15A-D, and 16B-D, respectively. The precursorions selected in Q1 and the collision energies used to generate theseproduct ion spectra are indicated in Table 16.

Exemplary MRM transitions for the quantitation ofPTAD-1α,25-hydroxyvitamin D₃ include fragmenting a precursor ion with am/z of about 538.4 to a product ion with a m/z of about 278.1;fragmenting a precursor ion with a m/z of about 556.4 to a product ionwith a m/z of about 298.0; and fragmenting a precursor ion with a m/z ofabout 574.4 to a product ion with a m/z of about 313.0. Exemplary MRMtransitions for the quantitation of PTAD-1α,25-dihydroxyvitamin D₃-[6,19, 19]-²H₃ include fragmenting a precursor ion with a m/z of about541.4 to a product ion with a m/z of about 280.9; fragmenting aprecursor ion with a m/z of about 559.4 to a product ion with a m/z ofabout 301.1; and fragmenting a precursor ion with a m/z of about 577.4to a product ion with a m/z of about 317.3. Exemplary MRM transitionsfor the quantitation of PTAD-1α,25-dihydroxyvitamin D₂-[26, 26, 26, 27,27, 27]-²H₆ include fragmenting a precursor ion with a m/z of about544.4 to a product ion with a m/z of about 278.0; fragmenting aprecursor ion with a m/z of about 562.4 to a product ion with a m/z ofabout 298.2; and fragmenting a precursor ion with a m/z of about 580.4to a product ion with a m/z of about 314.0. However, as can be seen inthe product ion scans in FIGS. 14B-D, 15B-D, and 25B-D, several otherproduct ions are generated upon fragmentation of the precursor ions.Additional product ions may be selected from those indicated in FIGS.14B-D, 15B-D, and 16B-D to replace or augment the exemplary fragmentions.

TABLE 16 Precursor Ions and Collision Cell Energies for Fragmentation ofPTAD-1α,25-dihydroxyvitamin D₃, PTAD-1α,25-dihydroxyvitamin D₃-[6, 19,19]-²H₃, and PTAD-1α,25-dihydroxyvitamin D₃- [26, 26, 26, 27, 27,27]-²H₆ Collision Precursor Cell Energy Analyte Ion (m/z) (V)PTAD-1α,25-dihydroxyvitamin D₃ 538.4, 556.4, 574.4 15PTAD-1α,25-dihydroxyvitamin 541.4, 559.4, 577.4 15 D₃-[6,19,19]-²H₃PTAD-1α,25-dihydroxyvitamin 544.4, 562.4, 580.4 15D₃-[26,26,26,27,27,27]-²H₆

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining an amount of oneor more vitamin D metabolites in a sample by mass spectrometry, themethod comprising: (i) subjecting the sample and an internal standard toa Cookson-type derivatizing reagent under conditions sufficient togenerate one or more Cookson-type vitamin D metabolite derivatives andone or more Cookson-type internal standard derivatives, wherein theinternal standard comprises at least one of 25OHD₂-[6, 19, 19]-²H₃,25OHD₂-[26, 26, 26, 27, 27, 27]-²H₆, 25OHD₃[6, 19, 19]-²H₃, 25OHD₃-[26,26, 26, 27, 27, 27]-²H₆, 1α,25(OH)₂D₃-[6, 19, 19]-²H₃, 1α,25(OH)₂D₂-[26,26, 26, 27, 27, 27]-²H₆, 1α,25(OH)₂D₃-[6, 19, 19]-²H₃ and1α,25(OH)₂D₂-[6, 26, 26, 27, 27, 27]-²H₆; (ii) subjecting the one ormore Cookson-type vitamin D metabolite derivatives and the one or moreCookson-type internal standard derivatives to turbulent flow liquidchromatography (TFLC); (iii) ionizing the one or more Cookson-typevitamin D metabolite derivatives and the one or more Cookson-typeinternal standard derivative to generate respectively one or moreCookson-type vitamin D metabolite derivative ions and one or moreCookson-type internal standard derivative ions detectable by massspectrometry; (iv) determining an amount of the one or more of theCookson-type vitamin D metabolite derivative ions and an amount of theone or more Cookson-type internal standard derivative ions by massspectrometry; and (v) relating the amounts of the Cookson-type vitamin Dmetabolite derivative ions and the one or more Cookson-type internalstandard derivative ions determined in (iv) to the amount of a vitamin Dmetabolite in the sample.
 2. The method of claim 1, wherein the sampleis subjected to high performance liquid chromatography (HPLC) after (ii)but prior to (iii).
 3. The method of claim 2, wherein the TFLC of (ii),the HPLC, and the ionization of (iii) are conducted in an on-linefashion.
 4. The method of claim 1, wherein the Cookson-type derivatizingreagent comprises at least one of 4-phenyl-1,2,4-triazoline-3,5-dione(PTAD),4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQTAD), 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD), and4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), and isotopicallylabeled variants thereof.
 5. The method of claim 1, wherein the massspectrometry is tandem mass spectrometry.
 6. The method of claim 1,wherein the one or more vitamin D metabolites comprise at least one of1α,25-dihydroxyvitamin D₂ (1α,25(OH)₂D₂) and 1α,25-dihydroxyvitamin D₃(1α,25(OH)₂D₃).
 7. The method of claim 1, wherein the one or moreCookson-type vitamin D metabolite derivative ions comprise ions selectedfrom the group consisting of PTAD-25OHD₂ ions with a m/z ratio of570.3±0.5 and 298.1±0.5.
 8. The method of claim 1, wherein the one ormore Cookson-type vitamin D metabolite derivative ions comprise at leastone PTAD-25OHD₂ parent ion with a m/z ratio of 570.3±0.5 and at leastone PTAD-25OHD₂ fragment ion with a m/z ratio of 298.1±0.5.
 9. Themethod of claim 1, wherein the one or more Cookson-type vitamin Dmetabolite derivative ions comprise ions selected from the groupconsisting of PTAD-25OHD₃ ions with a m/z ratio of 558.3±0.5 and298.1±0.5.
 10. The method of claim 1, wherein the one or moreCookson-type vitamin D metabolite derivative ions comprise at least onePTAD-25OHD₃ parent ion with a m/z ratio of 558.3±0.5 and at least onePTAD-25OHD₃ fragment ion with a m/z ratio of 298.1±0.5.
 11. The methodof claim 1, wherein the internal standard is 25OHD₂-[6, 19, 19]-²H₃. 12.The method of claim 1, wherein the internal standard is 25OHD₃[6, 19,19]-²H₃.
 13. The method of claim 1, wherein the internal standard is1α,25(OH)₂D₃-[6, 19, 19]-²H₃.
 14. The method of claim 1, wherein theinternal standard is 1α,25(OH)₂D₃-[6, 19, 19]-²H₃.
 15. The method ofclaim 1, wherein the one or more vitamin D metabolites and the internalstandard are extracted with ethyl acetate.
 16. The method of claim 1,further comprising acetonitrile precipitation.
 17. The method of claim1, further comprising purification by solid phase extraction (SPE). 18.A method for determining an amount of one or more vitamin D metabolitesin a sample by mass spectrometry, the method comprising: (i) subjectingthe sample and an internal standard to4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) under conditions sufficientto generate one or more PTAD-derivatized vitamin D metabolites and oneor more PTAD-derivatized internal standard derivatives, wherein theinternal standard comprises at least one of 25OHD₂-[6, 19, 19]-²H₃25OHD₂-[26, 26, 26, 27, 27, 27]-²H₆, 25OHD₃[6, 19, 19]-²H₃, 25OHD₃-[26,26, 26, 27, 27, 27]-²H₆, 1α,25(OH)₂D₃-[6, 19, 19]-²H₃, 1α,25(OH)₂D₂-[26,26, 26, 27, 27, 27]-²H₆, 1α,25(OH)₂D₃-[6, 19, 19]-²H₃ and1α,25(OH)₂D₂-[6, 26, 26, 27, 27, 27]-²H₆; (ii) subjecting the one ormore PTAD-derivatized vitamin D metabolites in the sample and the one ormore PTAD-derivatized internal standard derivatives to a turbulent flowliquid chromatography (TFLC) column and an analytical column; (iii)subjecting the one or more PTAD-derivatized vitamin D metabolites in thesample and one or more PTAD-derivatized internal standard derivatives toan ionization source to generate one or more PTAD-derivatized vitamin Dmetabolite ions and one or more PTAD-derivatized internal standardderivative ions detectable by mass spectrometry; (iv) determining anamount of one or more of the PTAD-derivatized vitamin D metabolite ionsand an amount of the one or more PTAD-derivatized internal standardderivative ions by mass spectrometry; and (v) relating the amount ofPTAD-derivatized vitamin D metabolite ions and the amount of the one ormore PTAD-derivatized internal standard derivative ions determined in(iv) to the amount of the one or more vitamin D metabolites in thesample.
 19. The method of claim 18, wherein the analytical column of(ii) is a high performance liquid chromatography (HPLC) column.
 20. Themethod of claim 18, wherein the TFLC and analytical columns of (ii) andthe ionization source of step (iii) are connected in an on-line fashion.21. The method of claim 18, wherein the mass spectrometry is tandem massspectrometry.
 22. The method of claim 18, wherein the one or morePTAD-derivatized vitamin D metabolite ions comprise ions selected fromthe group consisting of PTAD-25OHD₂ ions with a m/z ratio of 570.3±0.5and 298.1±0.5.
 23. The method of claim 18, wherein the one or morePTAD-derivatized vitamin D metabolite ions comprise ions selected fromthe group consisting of PTAD-25OHD₃ ions with a m/z ratio of 558.3±0.5and 298.1±0.5.
 24. The method of claim 18, wherein the internal standardis 25OHD₂-[6, 19, 19]-²H₃.
 25. The method of claim 18, wherein theinternal standard is 25OHD₃[6, 19, 19]-2H₃.
 26. The method of claim 18,wherein the internal standard is 1α,25(OH)₂D₃-[6, 19, 19]-²H₃.
 27. Themethod of claim 18, wherein the internal standard is 1α,25(OH)₂D₃-[6,19, 19]-²H₃.
 28. The method of claim 18, wherein the one or more vitaminD metabolites and the internal standard are extracted with ethylacetate.
 29. The method of claim 18, further comprising acetonitrileprecipitation.
 30. The method of claim 18, further comprisingpurification by solid phase extraction (SPE).